Molecular biology is the study of biology at a molecular level.

1. Theme: Molecular basis of heredity. Realization of hereditary informationLecturer: ass. prof. Tetyana Bihunyak

2. The questions of the lecture:1. Molecular biology as science 2.  The chemistry of nucleic acids2.1. Deoxyribonucleic acid (DNA) 2.2. Ribonucleic acid (RNA) 3. DNA Replication 4. Genetic code5. Gene Expression6. Functions of Proteins in the organism

3. Molecular biology is the study of biology at a molecular level. Molecular biology concerns itself with understanding the interactions between the various systems of a cell, including the interactions between DNA, RNA andprotein biosynthesis and learning how these interactions are regulated.

4. Nucleus contains genetic materials encoded in DNA of chromosomes Only nucleus directs protein synthesis in the cytoplasm via ribosomal RNA (rRNA), messenger RNA (mRNA) and transport RNA (tRNA), which are synthesized in the nucleus

5. Nucleic acids The nucleic acids are polymers of smaller units called nucleotides. There are 2 types of nucleic acids: DNA (deoxyribonucleic acid) RNA (ribonucleic acid).

6. Structure of nucleotide • five-carbon sugar (deoxyribose C5H10O4 in DNA and ribose C5H10O5 in RNA); • a phosphate group (PO4); • one of five types nitrogen-containing compounds called nitrogenous bases.

7. Nucleic acids The nitrogenous bases are: Purines, which are larger – Adenine (A), Guanine (G); Pyramidines, which are smaller – Thymine (T), Cytosine (C), Uracil (U).

8. DNA located in nucleus, packaged into chromosomes Chromosome DNA Nucleus Nucleotides

9. DNA Basics Organelles (mitochondria, chloroplasts) have their own chromosomes (DNA)

10. DNA Basics • DNA is a long, double-stranded, linear molecule composed of multiple nucleotide sequences. • DNA contains Adenine, Guanine, Cytosine, Thymine.

11. Nitrogenous bases Purines Pyrimidines Thymine (T) Cytosine (C)

12. DNA Basics The DNA double helix consists of two complementary DNA strands held together by hydrogen bonds between the base pairs A-T and G-C

13. A-T base pair Hydrogen bonding of the bases G-C base pair Chargaff’s rule: The content of A equals the content of T, and the content of G equals the content of C in double-stranded DNA from any species

14. Chargaff's rules said that A = T and G = C. The model shows that A is hydrogen bonded to T and G is hydrogen bonded to C. This so-called complementary base pairing means that a purine is always bonded to a pyrimidine. Only in this way will the molecule have the width (2 nm) dictated by its X-ray diffraction pattern, since 2 pyrimidines together are too narrow and 2 purines together are too wide.

15. DNA Basics In the formation of a nucleic acid chain the phosphate group of the nucleotide binds to the hydroxyl group of another, forming what is called a phosphodiester bond, which is very strong.

16. Crick Francis Watson James The double helix of DNA was discovered in 1953 by Crick F. and Watson J. Nobel prize in 1962

17. Structure of DNA Watson and Crick model shows that DNA is a double helix with sugar-phosphate backbones on the outside and paired bases on the inside. This arrangement first the mathematical measurements provided by the X-ray diffraction data for the spacing between the base pairs (0.34 nm) and for a complete turn of the double helix (3.4 nm)

19. The main biological DNA functions: • DNA stores hereditary information about primary protein structure. • The order of the bases specifies the order of amino acids in polypeptides. • DNA-replication – maintaining genetic information.

20. DNA Replication • The Watson and Crick model suggests that DNA can be replicated by means of complementary base pairing. • During replication, each old DNA strand of the parent molecule serves as a template for a new strand in a daughter molecule. • A template is most often a mold used to produce a shape complementary to itself

21. S phase is the synthetic phase, resulting in duplication of the chromosomes: one replicated chromosome consisting of two chromatids Thehuman cell cycle Rapid growth and preparation for DNA synthesis S phase G0 G1 phase Quiescent cells G2 phase Growth and preparation for cell division M phase Mitosis

22. Steps of replication: • 1. Unwinding • The old strands that make up the parent DNA molecule are unwound and "unzipped" (i.e., the weak hydrogen bonds between the paired bases are broken) • There is a special enzyme called helicase that unwinds the molecule Helicase

23. DNA replication • DNA helicase (enzyme) unwinds the DNA. The junction between the unwound part and the open part is called a replication fork. • DNA polymerase adds the complementary nucleotides and binds the sugars and phosphates. DNA polymerase travels from the 3' to the 5' end.

24. Steps of replication: • 2. Complementary base pairing • New complementary nucleotides, • always present in the nucleus, • are positioned by the process • of complementary base pairing.

25. DNA replication • DNA polymerase adds complementary nucleotides on the other side of the ladder. Traveling in the opposite direction. • One side is the leading strand - it follows the helicase as it unwinds. • The other side is the lagging strand - its moving away from the helicase

26. DNA replication • Problem: it reaches the replication fork, but the helicase is moving in the opposite direction. It stops, and another polymerase binds farther down the chain. • This process creates several fragments, called Okazaki Fragments, that are bound together by DNA ligase.

27. Steps of replication: • 3. Joining • The complementary nucleotides become joined together to form new strands. • Each daughter DNA molecule contains an old strand and a new strand. • Steps 2 and 3 are carried out by the enzyme DNA polymerase.

28. DNA replication • During replication, there are many points along the DNA that are synthesized at the same time (multiple replication forks). • It would take forever to go from one end to the other, it is more efficient to open up several points at one time.

29. A model for DNA replication DNA replication is termed semiconservative replicationbecause one of the old strands is conserved, or present, in each daughter double helix. Semiconservative replication was experimentally confirmed by Matthew Meselson and Franklin Stahl in 1958.

30. Accuracy of Replication • The mismatched nucleotide causes a pause in replication, and during this time, the mismatched nucleotide is excised from the daughter strand. • The errors that slip through nucleotide selection and proofreading cause a gene mutation to occur. • Actually it is of benefit for mutations to occur occasionally because variation is the raw material for the evolutionary process.

31. Rate of Gene Mutations • Per cell cycle, gene mutations don't occur very often • There are several mechanisms that protect against the occurrence of mutations. • The bases are on the interior of the DNA molecule, and the supercoiling of the molecule in eukaryotes also lends stability. • During replication, DNA polymerases proofread the new strand against the old strand and detect any mismatched pairs, which are then replaced with the correct nucleotides. In the end, there is usually only one mistake for every one billion nucleotide pairs replicated.

32. deamination ATGCUGCATTGA TACGGCGTAACT uracil DNA glycosylase ATGCGCATTGA TACGGCGTAACT repair nucleases AT GCATTGA TACGGCGTAACT DNA polymerase b ATGCCGCATTGA TACGGCGTAACT DNA ligase ATGCCGCATTGA TACGGCGTAACT Excision repair thymine dimer ATGCUGCATTGATAG TACGGCGTAACTATC excinuclease AT AG TACGGCGTAACTATC (~30 nucleotides) DNA polymerase b ATGCCGCATTGATAG TACGGCGTAACTATC DNA ligase ATGCCGCATTGATAG TACGGCGTAACTATC Base excision repair Nucleotide excision repair

33. Correlation between DNA repair activity in fibroblast cells from various mammalian species and the life span of the organism 100 human elephant cow Life span 10 hamster rat mouse shrew 1 DNA repair activity

34. Point Mutations • Point mutations involve a change in a single nucleotide and therefore a change in a specific codon. • When one base is substituted for another, the results can be variable. For example, if UAC is changed to UAU, there is no noticeable effect, because both of these codons code for tyrosine.This is called a silent mutation. • If UAC is changed to UAG, however, the result could very well be a drastic one because UAG is a stop codon. If this substitution occurs early in the gene, the resulting protein may be too short and may be unable to function. Such an effect is called a nonsense mutation. Finally, if UAC is changed to CAC, then histidine is incorporated into the protein instead of tyrosine. This is a missense mutation. • A change in one amino acid may not have an effect if the change occurs in a noncritical area or if the 2 amino acids have the same chemical properties. In this instance, the polarities of tyrosine and histidine differ; this substitution most likely will have a deleterious effect on the functioning of the protein. Recall that the occurrence of valine instead of glutamate in the beta (B) chain of hemoglobin results in asickle-cell disease.

35. Defects in DNA repair or replication • All are associated with a high frequency of chromosome • and gene (base pair) mutations; most are also associated with a • predisposition to cancer, particularly leukemias • Xeroderma pigmentosum • caused by mutations in genes involved in nucleotide excision repair • associated with a >1000-fold increase of sunlight-induced • skin cancer and with other types of cancer such as melanoma • Ataxia telangiectasia • caused by gene that detects DNA damage • increased risk of X-ray • associated with increased breast cancer in carriers • Fanconi anemia • caused by a gene involved in DNA repair • increased risk of X-ray and sensitivity to sunlight • Bloom syndrome • caused by mutations in a DNA helicase gene • increased risk of X-ray • sensitivity to sunlight • Cockayne syndrome • caused by a defect in transcription-linked DNA repair • sensitivity to sunlight • Werner’s syndrome • caused by mutations in a DNA helicase gene • premature aging

36. DNA and RNA differ • RNA is single-stranded (but it can fold back upon itself to form secondary structure, e.g. tRNA) • In RNA, the sugar molecule is ribose rather than deoxyribose • In RNA, the fourth base is uracil rather than thymine.

37. RNA 3 U H OH OH OH OH 1 2 DNA

38. The major bases found in DNA and RNA DNA RNA Adenine Adenine Cytosine Cytosine Guanine Guanine Thymine Uracil (U) thymine-adenine base pair uracil-adenine base pair

39. RNA Basics Messenger RNA carries the genetic code to the cytoplasm to direct protein synthesis. 1. This single-stranded molecule (hundreds to thousands of nucleotides). 2. mRNA contains codons that are complementary to the DNA codons from which it was transcribed Ribosomal RNA associates with many different proteins (including enzymes) to form ribosomes. 1. rRNA associates with mRNA and tRNA during protein synthesis. 2. rRNA synthesis takes place in the nucleolus and is catalyzed by RNA polymerase.

40. Transfer RNA - the adapter Transfer RNA is folded into a cloverleaf shape and contains about 80 nucleotides. • 1. Each tRNA combines with a specific amino acid that has been activated by an enzyme. • 2. One end of the tRNA molecule possesses an anticodon, a triplet of nucleotides that recognizes the complementary codon in mRNA.

41. Types of RNA

42. Transcription: DNA-Directed RNA Synthesis • Transcription has three phases: • Initiation • Elongation • Termination • RNA is transcribed from a DNA template after the bases of DNA are exposed by unwinding of the double helix. • In a given region of DNA, only one of the two strands can act as a template for transcription.

43. Figure 12.4 – Part 1

44. Transcription: DNA-Directed RNA Synthesis - Elongation • Nucleotides are added by complementary base pairing with the template strand • The substrates, ribonucleoside triphosphates, are hydrolyzed as added, releasing energy for RNA synthesis.

45. DNA RNA RNA U U OH OH OH OH OH OH OH OH OH OH DNA Replication figure adapted for Transcription

46. All organisms use the same genetic codeEach set of three nucleotides codes for an amino acid = “The Genetic Code” Genetic code

47. The Genetic Code is universal • All organisms use the same genetic code • Each set of three nucleotides codes for an amino acid = “The Genetic Code” AUG = Met

48. The genetic code consists of 64 triplet codons (A, G, C, U) 43 = 64 all codons are used in protein synthesis 20 amino acids 3 termination (stop) codons: UAA, UAG, UGA AUG (methionine) is the start codon (also used internally) multiple codons for a single amino acid = degeneracy Genetic code is unambiguous. Each triplet codon has only one meaning 5 amino acids are specified by the first two nucleotides only 3 additional amino acids (Arg, Leu, and Ser) are specified by six different codons

49. Gene Expression • The process by which a gene produces a product, usually a protein, is calledgene expression. • DNA not only serves as a template for its own replication, it is also a template for RNA formation. • Gene Expression in prokaryotes: • transcription, translation. • Gene Expression in eukaryotic cells: • transcription, processing, translation.

50. Transcription • The process by which a mRNA copy is made of a portion of DNA • It is the first step required for gene expression. • During transcription, a mRNA molecule is formed that has a sequence of bases complementary to a portion of one DNA strand; • A, T, G, or Сis present in the DNA template, • U, A, C, or G is incorporated into the mRNA molecule